Division 1.9.2 Performance of the test

About 50 to 200 g of soil (dry weight basis) are placed into each incubation flask (see Figures 1 and 2 in Annex 3) and the soil treated with the test substance by one of the methods described in section 1.8.2. When organic solvents are used for the application of the test substance, they should be removed from soil by evaporation. Then the soil is thoroughly mixed with a spatula and/or by shaking of the flask. If the study is conducted under paddy field conditions, soil and water should be thoroughly mixed after application of the test substance. Small aliquots (e.g. 1 g) of the treated soils should be analysed for the test substance to check for uniform distribution. For alternative method, see below.

The treatment rate should correspond to the highest application rate of a crop protection product recommended in the use instructions and uniform incorporation to an appropriate depth in the field (e.g. top 10 cm layer^f1 of soil). For example, for chemicals foliarly or soil applied without incorporation, the appropriate depth for computing how much chemical should be added to each flask is 2.5 cm. For soil incorporated chemicals, the appropriate depth is the incorporation depth specified in the use instructions. For general chemicals, the application rate should be estimated based on the most relevant route of entry; for example, when the major route of entry in soil is through sewage sludge, the chemical should be dosed into the sludge at a concentration that reflects the expected sludge concentration and the amount of sludge added to the soil should reflect normal sludge loading to agricultural soils. If this concentration is not high enough to identify major transformation products, incubation of separate soil samples containing higher rates may be helpful, but excessive rates influencing soil microbial functions should be avoided (see sections 1.5 and 1.8.2).

Alternatively, a larger batch (i.e. 1 to 2 kg) of soil can be treated with the test substance, carefully mixed in an appropriate mixing machine and then transferred in small portions of 50 to 200 g into the incubation flasks (for example with the use of sample splitters). Small aliquots (e.g. 1 g) of the treated soil batch should be analysed for the test substance to check for uniform distribution. Such a procedure is preferred since it allows for more uniform distribution of the test substance into the soil.

Also untreated soil samples are incubated under the same conditions (aerobic) as the samples treated with the test substance. These samples are used for biomass measurements during and at the end of the studies.

When the test substance is applied to the soil dissolved in organic solvent(s), soil samples treated with the same amount of solvent(s) are incubated under the same conditions (aerobic) as the samples treated with the test substance. These samples are used for biomass measurements initially, during and at the end of the studies to check for effects of the solvent(s) on microbial biomass.

The flasks containing the treated soil are either attached to the flow-through system described in Figure 1 or closed with the absorption column shown in Figure 2 (see Annex 3).